Dimensiones del modelo sociocultural de estrés y afrontamiento adaptado al

Normal primary human umbilical vein endothelial cells (HUVECs; Pooled, PCS-100–013) were obtained from ATCC (Manassas, VA, USA) and grown at 37°C and 5% CO2 on uncoated T75 flasks in vascular cell basal medium (ATCC, PCS-100–030) containing 10% fetal bovine serum (FBS), VEGF endothelial cell growth kit (ATCC, PCS-100–041) and treated with DEX using 2%

FBS (both medium were prepared without hydrocortisone hemisuccinate), 100 units/mL penicillin and 100 μg/mL streptomycin. Cells were kept frozen in medium containing 10% DMSO (Bio-Rad Laboratories Canada Ltd., Mississauga, ON, Canada). For experiments, passage 3 was used. Cells were counted using a haemocytometer and seeded onto permeable polyethylene terephthalate (PET) filters at the base of BD Falcon cell culture inserts (BD Biosciences, Mississauga, ON, Canada) at a density of 0.5 × 10⁶ cells/insert.

Dexamethasone (DEX) was obtained from Sigma-Aldrich (Oakville, ON, Canada), and full- length Ad was produced in-house using a mammalian expression system (i.e. HEK 293 cells) according to methods described by (288, 303). HUVECs were treated for 5 days with DEX (1 μM) starting 24 h after seeding cells into inserts, HUVECs were treated with DEX added to both apical and basolateral sides of inserts at concentrations indicated above

2.3.2. shRNA-mediated knockdown of claudin 7 in HUVEC

We used pGPU6/Neo-claudin-7 shRNA vector with target sequence (5’- GGCCATCAGATTGTCACAGAC-3’) (GenePharma Co., Ltd., Shanghai, China). These were transfected into HUVEC using Lipofectamine™ 3000 Reagent (Invitrogen, Carlsbad, CA, USA) exactly according to manufacturer’s protocol. Non-specific scrambed target sequence shRNA vector was used as control. Following selection with 50 μg/ml Neomycin (Sigma-Aldrich, Oakville, ON,

CA) for 24 hours, cells were seeded onto inserts for analysis of TEER, examining adiponectin flux or preparation of cell lysates to confirm claudin 7 knock-down.

2.3.3. Transendothelial electrical resistance (TEER),and [³H]PEG4000 and Adflux

TEER was measured daily using chopstick electrodes (STX-2) connected to an EVOM voltohmmeter (World Precision Instruments, Sarasota, FL, USA). As a measure of paracellular permeability, apical to basolateral flux rates of [³H] polyethylene glycol at 1 μCi; 1 h flux (molecular mass 4000 Da; PEG-4000; PerkinElmer, Woodbridge, ON, Canada) or Ad (10 μg/mL; 24 h flux) to apical culture medium were determined across HUVEC endothelia. [³H] PEG-4000 in basolateral culture medium was detected using a liquid scintillation counter, Ad was detected using a mouse Ad ELISA kit (Antibody Immunoassay Services, Hong Kong) or Western blot. Permeability measurements were expressed according to calculations previously outlined by (300).

2.3.4. L6 and H9C2 cell culture and treatment with HUVEC-conditioned medium

Rat myoblasts and cardiomyocytes at passage 22–30 were grown in α_AMEM(Life Technologies Inc., Manassas, MA, USA) and DMEM containing 10% FBS and 1% antibiotic-antimycotic (Wisent Inc., St- Bruno QC, Canada). Culture media from the basolateral compartment of HUVEC inserts were collected and applied. L6/ H9C2 cells were starved with medium containing 0.5% FBS and 1% antibiotic-antimycotic for 3 h prior to treatment with HUVEC-conditioned medium.

2.3.5. Quantitative real-time PCR analysis

Total RNA was isolated from control and DEX-treated HUVECs and from soleus skeletal muscle using TRIzol Reagent . Extracted RNA was then treated with DNase I and first- strand cDNA was synthesized using SuperScript III reverse transcriptase and oligo(dT)12–18 primers (Life

Technologies Inc.,Manassas MA, USA). Quantitative real-time PCR (qRT- PCR) analyses were conducted using gene specific primers. SYBR Green I Supermix (Bio- Rad Laboratories Canada Ltd.Mississauga, ON, Canada) and a Chromo4 Detection System (CFB-3240; Bio-Rad Laboratories Canada Ltd.) Samples were run in duplicate. For all qRT- PCR analyses, TJ protein mRNA expression was normalized to GAPDH transcript abundance. For the expression profile, TJ protein transcripts were expressed relative to occludin mRNA. For TJ protein transcripts that were not detected in HUVECs, normal human adult kidney cDNA was obtained (BioChain Institute, Inc., Newark, CA, USA) and used as a positive control in qRT-PCR reactions. Agarose gel electrophoresis verified single qRT-PCR products at predicted amplicon sizes from positive control reactions.

2.3.6. Animal model of diabetes induced by exogenous corticosterone treatment

We used a well-established hyperinsulinemic/hyperglycemic rodent model of chronic glucocorticoid treatment (19, 20, 53, 241, 244). Upon experiment cessation, the tibialis anterior (TA) and soleus skeletal muscles were excised and immediately frozen in liquid N2 and kept at −80^oC until future analysis.

2.3.7. Oral glucose tolerance test

Animals were fasted overnight (16 hours), 11 days after pellet implantation, and were administered an oral glucose tolerance test (OGTT, 1.5 g/kg body mass) on day 12 using glucometer (Bayer One Touch), additional fasted plasma was collected for later analysis of insulin concentrations via an ELISA 96-well kit (Crystal Chem, USA). Plasma for measurement of Ad concentration was obtained 7 days after pellet implantation at 0800 h and assessed using a mouse Ad ELISA kit (AIS, Hong Kong).

2.3.8. Measurement of hydraulic conductivity (Lp) in individually perfused rat mesenteric microvessel

Female Sprague-Dawley rats of 220 to 250 g (2 to 3 mo old, Sage Laboratory Animal, PA) were used for the experiments. All procedures and animal use were approved by the Animal Care and Use Committee at Pennsylvania State University. Inactin hydrate (Sigma) was used for anesthesia and given subcutaneously at 170 mg/kg body weight. Microvessel permeability was assessed by measuring Lp in individually perfused microvessels, which measures the volume of water flux across the microvessel wall. Details have been described previously (318, 319).

2.3.9. Staining for metachromatic myosin ATPase

To identify skeletal muscle fiber type, a metachromatic myosin ATPase stain was performed using a modified protocol (Ogilvie and Feeback 1990). Sections were pre-incubated in an acidic buffer (pH=4.25) to differentially inhibit myosin ATPase within the different fiber types. In this protocol, type I fibers appear dark blue, type IIa appear light blue and type IIb and IIx are not apparent from each other and are classfied as IIb/x. These fibers appear almost white and are the largest.

Images were acquired with a Nikon Eclipse 90i microscope and imaging MicroPublisher with Q-Capture software at 10x magnification.

2.3.10. Immunohistochemistry of Ad and dystrophin in skeletal muscle

Tibialis anterior (TA) from Control and CORT treated rats were cryostat sectioned (10 µm thick) for analysis of muscle Ad content. Sections were stained as previously described (139).

Quantification was performed using Zen 2.0 software. The total Ad and dystrophin signal was determined by the sum of red/green signal intensity obtained arbitrary values in the field of view.

Diagram view intensity was recorded by the software in form of histograms. Intracellular

quantification was done on Ad images (without dystrophin), images were changed to 8-bits on Image and the arbitrary intensity was calculated as mean intensity per area.

2.3.11. Western blot analysis

Control and DEX-treated HUVECs, and L6 or H9C2 cells treated with HUVEC-conditioned medium and soleus skeletal muscle were lysed in sample buffer (80 mM Tris-HCl (pH 6.8), 2% (w/v) SDS, 20% glycerol, 3.3% (v/v) β-mercaptoethanol 0.01% w/v bromophenol blue) containing protease and phosphatase inhibitors (3 mM EDTA, 10 uM E64, 1 mM Na3VO4, 1 μM leupeptin, 1 μM pepstatin A, 1 μM okadaic acid and 200 μM PMSF). Apical and basolateral HUVEC-conditioned media collected from Ad flux experiments were concentrated with Amicon Ultra-4 Centrifugal Filter Units with Ultracel-30 membranes (EMD Millipore, Billerica, MA, USA) and subjected to nondenaturing, nonreducing conditions to allow the analysis of the different forms of Ad (HMW

>250 kDa, MMW ~180 kDa, and LMW ~90 kDa). Primary antibodies specific for the following proteins: T-cadherin (1:1000, R&D Systems), occludin (OCLN, 1:3000), tricellulin (TRIC, 1:3000), claudin-7 (CLDN-7, 1:500), CLDN-10 (1:800), CLDN-11 (1:1000), phospho-AMPKα (Thr172) (1:1000), phospho-p38 MAPK [pT180/pY182] (1:1000), Ad (1:1000),β-actin (1:1000).

OCLN, TRIC, CLDN-7, CLDN-10 and phospho-p38 MAPK [pT180/pY182] antibodies were obtained from Life Technologies, CLDN-11 and Ad antibodies were purchased from EMD Millipore and Signalway Antibody (College Park, MD, USA) respectively, and phospho-AMPKα (Thr172) and β-actin antibodies were obtained from Cell Signaling Technology (New England Biolabs Ltd., Whitby, ON, CA). Protein detection using enhanced chemiluminescence (Bio-Rad) and quantification by densitometry using ImageJ analysis software. TJ protein expression was normalized to β-actin, Tubulin protein abundance.

2.3.12. Statistical analysis

All data are expressed as mean values ± SEM. A one-way analysis of variance (ANOVA) followed by a Student-Newman-Keuls test was used to determine significant differences (P ≤ 0.05) between groups. When appropriate, a Student’s t-test was also used. All statistical analyses were conducted using Prism 5, Excel SigmaStat 3.5 softwares.